Roxadustat

Roxadustat (FG-4592; Selleck Chemicals, Munich, Germany, #S1007) was dissolved in DMSO (10 mg/mL or 40 mg/mL) followed by dilution in 0.9% NaCl with 2% Tween-80 to a final concentration of 1 mg/mL or 4 mg/mL, respectively. Roxadustat was applied intraperitoneally (injection volume 0.2 mL per 20 g body weight) to 8-week-old male C57BL/6 mice at a dosage of either 10 mg/kg or 40 mg/kg body weight twice daily with 12 h between doses for 4 days. Control mice received equal volumes of vehicle solution (10% DMSO, 2% Tween-80 in 0.9% NaCl) twice a day for 4 days through intraperitoneal injection.
All mice were randomly allocated to experimental groups. Experimenters were blinded for animal treatment in all experiments and analyses. Evaluation of all read-out parameters was done independently and in a blinded fashion.

8-week-old wild type female mice received intraperitoneal (i.p.) injections of either vehicle (5%DMSO/45%PEG300/50%ddH2O) or 50 mg·kg⁻¹ of FG-4592 (‘FG’, Roxadustat; SelleckChem) or BAY 85-3934 (‘BAY’, Molidustat; SelleckChem) every other day for 5 days (3 injections total). Female flox-Fgf23/Dmp1-cre⁺ and cre⁻ mice (10-11 weeks old) were injected with 70 mg·kg⁻¹ of FG-4592 every other day for 5 days (3 injections total). In all studies, tissues were harvested 4 hours after the final injection.

The regulation of the WNT7A promoter mediated by HIF-1α was evaluated with the Nano-Glo^® Dual-Luciferase^® Assay System kit (Promega), which enables a high-throughput analysis of mammalian cells containing the following reporter genes: (a) Nanoluc (pNL1.1empty and pNL1.1WNT7A_promoter) and (b) pGL4.54(luc2/TK), containing the reporter gene Firefly as internal control. Briefly, cells were seeded in a 96-well plate at a concentration of 5 × 10³ cells per well, transfected with the pNL1.1 or pNL1.1WNT7A_promoter (90 ng) plasmids and pGL4.54(luc2/TK; 10 ng) plasmid as internal control, according to ViaFect Transfection Reagent^® protocol (Promega), and then incubated for 24 h. After transfection, cells were exposed for 24 h to hypoxic culture conditions (1% O₂) or to the PHDs inhibitors, IOX2 (Merck) or FG-4592 (Roxadustat, Selleckchem), at their respective IC₅₀ working concentrations. Then, an equal volume of ONE-Glo^TM EX Luciferase Assay Reagent was added to each well, incubated for 30 min, and then the luminescence was analyzed with a Varioskan^TM LUX Multimode Microplate Reader (Thermo Fisher Scientific). To turn off the luminescence of the Firefly luciferase and provide the substrate for the Nanoluc, an equal volume of NanoDLR^TM Stop&Glo^® Reagent was added to the mixture and incubated for 30 min before analysis.

Roxadustat (S1007), erastin (S7242), ferrostatin-1 (S7243), glutathione (S4606) and temozolomide (S1237) were from Selleck Chemicals (Texas, USA). BODIPY™ 581/591 C11 (D3861) was from Invitrogen, ThermoFisher (California, USA). Prussian Blue Iron Stain Kit (G1422) was from Solarbio Life Sciences (Beijing, China). Fluorescence labeled anti-Calreticulin (#62304) was from Cell Signaling Technology (CST). Primary antibodies were as follows: GPX4 (sc-166570, Santa Cruz), FTH1 (#3998 S, CST), HIF-1α (#36169 S, CST), β-actin (#4970 S, CST), HIF-2α (ab109616, abcam), 4-HNE (ab48506, abcam), Ki67 (sc-15402, Santa Cruz).

C57BL/6 mice were treated by intraperitoneal (i.p.) injection with roxadustat (33 mg/kg body weight in 0.5 M Tris-HCl, pH 9; Selleckchem, Munich, Germany) for seven or 14 days. Control mice were treated with the solvent without adding roxadustat. All animal work conformed to institutional guidelines by the Niedersächsische Landesamt für Verbraucherschutz und Lebensmittelsicherheit (approval number: 33.9-42502-04-14/1498).

Cell viability was determined using CCK-8 assays. Briefly, cells were seeded at a density of 5000 cells per well in 96-well plates and incubated for 24 h. Then, roxadustat (Selleck Chemicals, Houston, TX, USA, purity = 99.95%) was added to the medium at different concentrations. After roxadustat treatment for the indicated days, 10 µl CCK-8 reagent (DOJINDO, Tokyo, Japan) was added to each well, and the cells were incubated at 37 °C for 2 h. The absorbance at 450 nm was measured with a microplate reader (Bio-Rad, Model 680, USA).